Macrophage Survival Assay Protocol

Macrophage precursors were plated at 4 × 10⁴ cells/well in four separate CellCarrier-96 Ultra Microplates (Perkin Elmer), and differentiated in macrophage medium for 1 week. Survival was assessed as previously described^{23 (link)}. In brief, cells received a full media change to fresh macrophage medium with or without M-CSF (100 ng/ml), with triplicate wells per condition on each plate. One plate was used to assess the cell number at baseline (day 0), while the other plates were incubated at 37 °C/ 5% CO₂ for a further 3, 7, or 10 days. The 10-day plate received a 50% medium change at day 7. At the end of each incubation, cells were stained with the ReadyProbes Cell Viability Imaging Kit (Invitrogen) for 30 min at 37 °C/5% CO₂. Nuclei were imaged using the Opera Phenix High Content Screen System (Perkin Elmer) with a 10 × objective and 9 fields per well. Images were quantified with Columbus 2.7 software (Perkin Elmer). Data was presented as percentage of mean number of dead cells/mean number of total cells for each condition.